Hexahydrocannabinol (HHC) and Δ9-tetrahydrocannabinol (Δ9-THC) driven activation of cannabinoid receptor 1 results in biased intracellular signaling

The Cannabis sativa plant has been used for centuries as a recreational drug and more recently in the treatment of patients with neurological or psychiatric disorders. In many instances, treatment goals include relief from posttraumatic disorders, anxiety, or to support treatment of chronic pain. Ligands acting on cannabinoid receptor 1 (CB1R) are also potential targets for the treatment of other health conditions. Using an evidence-based approach, pharmacological investigation of CB1R agonists is timely, with the aim to provide chronically ill patients relief using well-defined and characterized compounds from cannabis. Hexahydrocannabinol (HHC), currently available over the counter in many countries to adults and even children, is of great interests to policy makers, legal administrators, and healthcare regulators, as well as pharmacologists. Herein, we studied the pharmacodynamics of HHC epimers, which activate CB1R. We compared their key CB1R-mediated signaling pathway activities and compared them to the pathways activated by Δ9-tetrahydrocannabinol (Δ9-THC). We provide evidence that activation of CB1R by HHC ligands is only broadly comparable to those mediated by Δ9-THC, and that both HHC epimers have unique properties. Together with the greater chemical stability of HHC compared to Δ9-THC, these molecules have a potential to become a part of modern medicine.


Synthesis and purification of HHC
Commercially-available CBD isolate (CBDepot, Czech Republic), p-toluenesulphonic acid (P-Lab, Czech Republic), and 5% palladium on activated charcoal (Merck KGaA, Germany) were used for the reaction.Solvents were purchased from a local distributor (Lach-Ner, Czech Republic) and were used without further purification.Solvents were evaporated using a vacuum rotary evaporator.Argon (5N) was used as an inert gas, and hydrogen (3.5N) was used for the reduction.Polar silica 40-63 µm (Merck KGaA, Germany) was used for ∆ 8 -THC purification.The Aldrich ® Kugelrohr™ short-path distillation apparatus (Merck KGaA, Germany) was used for HHC vacuum distillation.HHC epimers were separated using COMBIFLASH RF200 UV/VIS (Teledyne ISCO, United States) and RediSep Gold ® Silica Gel Disposable Flash Columns (Teledyne ISCO, United States).HPLC/ UV spectra were measured using LC/MS Agilent Technologies, 1290 Infinity DAD.The ratios of HHC epimers were determined based on signal characteristics in 1 H NMR spectra (δ 3.03 ppm for (9R)-HHC and δ 2.87 for (9S)-HHC).
The scale of the reaction ranged from 10 g of CBD up to 1 kg.CBD was dissolved in DCM to achieve a concentration of 50 g/L.For every gram of CBD, 0.5 g of p-toluenesulphonic was added.The mixture was flushed with argon and stirred for 48 h at room temperature.The reaction mixture was filtered through the silica column using 3 g of silica for every gram of CBD.The silica was washed with DCM until no more product was eluted.The solution of ∆ 8 -THC in DCM was concentrated to 1/10 of its original volume.An equal amount of MeOH was added diluting the solution approximately two times and the solution was evaporated once again to half of its volume.This procedure was repeated until no DCM signal (δ 5.30 ppm) was present on 1 H NMR. The resulting mixture of ∆ 8 -THC and MeOH was used for the reduction without further purification.
The corresponding conditions are listed in the Table 1.Palladium on activated charcoal was added to the solution of ∆ 8 -THC in MeOH.The reaction vessel was flushed with argon and then the argon was replaced by hydrogen.The mixture was stirred, and the pressure of hydrogen was maintained at around 1 atm.The mixture was filtered through celite and the celite was washed with MeOH until no more product was eluted.The MeOH was evaporated and the crude HHC was vacuum distilled using Kugelrohr™ (220 °C, 0.4 torr).A mixture of epimers (9R/S)-HHC (HPLC/UV purity 96%) was produced by this procedure.Samples of pure (9R)-HHC and (9S)-HHC were obtained from a 3:2 mixture (entry 2) using FLASH chromatography (hexane: t-BuOMe, 1-2%).

NMR characterization of HHC epimers
The NMR spectra were measured with Agilent 400 MR DDR2 (Agilent Technologies Inc., United States) using CDCl 3 (Merck KGaA, Germany) as a solvent and referenced on residual CDCl 3 signal ( 1 H δ 7.26 ppm).The spectra of corresponding epimers were identical to NMR spectra published by Russo et al. 11 .

Cell culture and transfection
Human Embryonic Kidney 293 (HEK293) cells (ATCC, USA, CRL-1573) were cultured in high glucose Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) supplemented with 10% fetal bovine serum (Gibco) at 37 °C, 5% CO 2 in the air, and 95% humidity.The cells were plated in 96-well plates (Greiner BioOne, UK) at 50,000 cells per well and transfected with 150 ng of DNA per well using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.The transfected cells were tested 24 h after transfection.

CB1R internalization assay
Cell surface receptor internalization was studied using the Homogenous Time-Resolved FRET (HTRF) technology as described previously 26 .First, HEK293 cells were seeded on a 96-well plate (Merck, Germany) and transiently transfected with SNAP-tagged CB1R plasmid together with empty vector pRK6 (1:2 DNA mass ratio) using Lipofectamine™ 2000 (Thermo Fisher Scientific) according to the manufacturer's protocol.Twenty-four hours post-transfection, the cell culture medium was removed, and the cells were labeled with 100 nM SNAP-Lumi4-Tb (PerkinElmer-CisBio, France), diluted in Tag-lite labeling medium (PerkinElmer-CisBio, France) and incubated for 1 h at 37 °C, 5% CO 2 .Subsequently, labeled cells were washed four times with Tag-Lite buffer solution.The receptor internalization experiment was performed by adding Tag-lite buffer containing 24 μM fluorescein (Merck, Germany) and corresponding CB1R agonist or vehicle (dimethyl sulfoxide, or in the case of Δ 9 -THC, ethanol).HTRF signal was recorded over 60 min at 37 °C using the Mithras LB 940 microplate reader (Berthold Technologies, Germany) equipped with the HTRF module and relevant filters.The donor fluorophore (terbium cryptate) was excited at 340 ± 26 nm and emission was measured at 520 ± 10 nm.The acceptor (fluorescein) emission was measured at 620 ± 10 nm.The HTRF ratio was calculated as the donor emission divided by the acceptor emission multiplied by 10,000.

Extracellular signal-regulated kinases 1/2 phosphorylation assay
Phosphorylation levels of endogenous extracellular signal-regulated kinases 1/2 (ERK1/2) were detected using the Phospho-ERK1/2 (Thr202/Tyr204) kit (Cisbio Bioassays, France).The transfected cells plated in 96-well plates (Greiner BioOne, UK) were serum-starved for 16 h prior to the experiment in serum-free DMEM media.Afterwards, the cells were stimulated for the indicated times by CB1R ligand diluted in serum-free DMEM and then lysed in 50 μl of supplemented lysis buffer.After homogenization, 16 μl of the cell lysate was transferred from the 96-well plate to a 384-well black plate (Greiner BioOne, UK) and incubated with 4 μl of detection buffer containing anti-ERK1/2-Eu 3+ cryptate and anti-Phospho-ERK1/2-d2 for at least 4 h in dark.The fluorescence emissions at 665 nm and 620 nm were read on HTRF® compatible Mithras LB 940 microplate reader (Berthold Technologies, Germany).Data are presented as the ratio of 665 nm emission and 620 nm emission multiplied by 10,000.
Because we used ∆ 8 -THC in the reduction reaction, we were able to confirm that, when a high concentration of ∆ 8 -THC and a relatively high amount of palladium on carbon is used, the reaction indeed predominantly produces (9R)-HHC at a ratio of approximately 3:1 (Table 1, entry 1).However, when only a small amount of www.nature.com/scientificreports/palladium on carbon and a low concentration was used, (9R)-HHC was produced in a lower ratio of 3:2 (entry 2).This reaction was also carried out in separate preparations at larger scales of 250 g (entry 3) and 1 kg (entry 4).For the scale-up reaction, a high concentration of ∆ 8 -THC and high amount of palladium on carbon was used, yielding predominantly (9R)-HHC in a 3:1 ratio.Achieving complete hydrogenation at large scales took significantly longer than at a small scale.Yields of HHC were not calculated as the hydrogenation reaction is quantitative and yields are mostly dependent on the scale of the reaction due to losses during distillation.

G protein activation induced by CB1R stimulation
To test whether HHC induces signaling via CB1R, we first measured the G protein activation in the transfected cells.We tested the effect of the studied cannabinoids (9S)-HHC, (9R)-HHC, Δ 9 -THC, and WIN on G protein activation by employing a BRET-based assay that monitors the dissociation of G α and G βγ subunits of the G i/o protein upon its activation by CB1R.We tested G i1 and G oA activation mediated by CB1R stimulation with increasing concentrations of (9S)-HHC, (9R)-HHC, Δ 9 -THC, and WIN.In all cases, agonist engagement of the receptor was followed by a prompt decrease in the BRET ratio, reflecting activation of the G proteins (Fig. 2).
In the G i1 and G oA activation assays, (9S)-HHC had a potency and efficacy lower than (9R)-HHC (Fig. 2 and Supplementary Tables 1 and 2).The potency and efficacy of (9R)-HHC were similar to those of Δ 9 -THC.Overall, the results demonstrate that the effect of (9R)-HHC epimer on the G i and G o signaling pathways is similar to that of Δ 9 -THC, while (9S)-HHC induces lower levels of the G protein activation.

GRK3 and β-arrestin2 interactions with the activated CB1R
We next studied the recruitment of GRK3 and β-arrestin2 to CB1R, as stimulated by the tested cannabinoids.The employed BRET-based interaction assays monitor the association of GRK3 and CB1R following receptor phosphorylation and the interaction of β-arrestin2 with the phosphorylated CB1R.Agonist activation of the receptor increased the BRET ratio, reflecting increased GRK3-CB1R and β-arrestin2-CB1R interactions (Fig. 3).
WIN application in these assays elicited the strongest responses and also showed the highest potency and efficacy (Fig. 3 and Supplementary Tables 3 and 4).On the other hand, the interactions induced by Δ 9 -THC were negligible.The potency of (9R)-HHC was higher than that of (9S)-HHC, but the curve fitting demonstrated that these epimers have similar efficacies.Overall, the results indicate that the (9R)-HHC epimer stimulates GRK3-CB1R and β-arrestin2-CB1R interactions more effectively than Δ 9 -THC or the (9S)-HHC epimer.

Internalization of activated CB1R
β-arrestin interaction with the desensitized receptor initiates receptor internalization and activates specific signaling cascades.We used the HTRF-based approach to monitor the kinetics of receptor internalization upon activation by the tested cannabinoids.In this approach, internalization results in increased emission of the terbium cryptate fluorophore that is covalently attached to the receptor.
Application of the cannabinoids initiated prompt and massive internalization of CB1R, but the extent of internalization varied.WIN had the highest effect on CB1R internalization rate (Fig. 4).The HHC epimers and Δ 9 -THC had comparable effects on CB1R internalization, which were about half of the WIN effect.  1 and 2.
Vol:.( 1234567890 G proteins and β-arrestin both contribute to the activity of the ERK1/2 signaling cascade.We determined the extent of ERK1/2 activity driven by the tested cannabinoids by measuring its phosphorylation in the HTRF-based sandwich ELISA.In this assay, ERK1/2 phosphorylation is detected as an increase in the HTRF ratio.WIN activation of CB1R led to a rapid but transient increase in ERK1/2 phosphorylation that peaked at 5 min after agonist stimulation and then progressively diminished (Fig. 5).Application of (9R)-HHC, (9S)-HHC, and Δ 9 -THC induced lower levels of ERK1/2 phosphorylation, peaking at 10 min.

Discussion
CB1R is the principal receptor of the central nervous system endocannabinoid system (ECS) 27 .CB1R is expressed in all brain regions, including those important for processing anxiety, fear, stress, and cognitive functions.CB1R is abundant in the basal ganglia, hippocampus, cerebellum, prefrontal cortex, and amygdala 28 .The neuronal ECS, with its central receptor, CB1R, is important for synaptic plasticity, strength, and maintenance.In addition to neurons, CB1R is also expressed in the central nervous system in astrocytes, microglia, and oligodendrocytes, where it modulates synaptic transmission, glucose metabolism, and immunomodulator production 18,29 .Furthermore, CB1R is also present in the peripheral nervous system, as well as in skeletal muscle, bone, skin, eyes, adipose tissue, and the reproductive system 30 .Subcellularly, CB1R is typically, but not exclusively, located presynaptically in many glutamatergic, GABAergic, cholinergic, serotonergic, and noradrenergic neurons.Endocannabinoids  3 and 4. are synthesized on demand on the postsynaptic side and suppress neurotransmitter release via activation of presynaptic CB1R [31][32][33] .CB1R is primarily directed to cell surface; however, an important discrete pool of CB1Rs is in the outer mitochondrial membrane 34 .
ECS is involved in appetite stimulation, energy balance regulation, learning and memory, pain processing, neurogenesis and neuroprotection, immune responses, and many other physiological regulations including neurohumoral system homeostasis.CB1R also plays an important role in pathological conditions including schizophrenia, multiple sclerosis, anxiety, depression, epilepsy, Parkinson's disease, Huntington's disease, Alzheimer's disease, addiction, stroke, inflammation, glaucoma, cancer, as well as musculoskeletal and liver disorders 16,35 .
The Cannabis sativa plant produces a vast repertoire of chemically and biologically interesting and diverse compounds.Over 400 compounds, about a quarter of which unique, have been detected in the plant.This remarkable mixture includes phytocannabinoids, terpenes and other compound classes 2,3 .Recent efforts to use a scientific approach to marijuana for medical purposes, namely in Canada, Israel, the USA, and the Czech Republic, have led to an approach in which two main substances, Δ 9 -THC and CBD, were evaluated by controlled trials in a broad cohort of patients with favorable outcomes.However, it is known that Cannabis sativa chemistry is not limited to only these two compounds, and many more structures must be taken into an account.
One historically-overlooked CB1R ligand, HHC, share a similar chemical structure with Δ 9 -THC and CBD.Herein we show that they activate CB1R in a unique way, most likely by favoring differential active conformational states of CB1R than those favored by Δ 9 -THC.Recent studies in mice have shown that HHC compounds are psychoactive, namely in the cannabinoid tetrad tests.Many results from behavioral analyses highlight generally overlapping, but not entirely parallel impacts, on the performance in the tests.The pharmacodynamic analyses presented here, together with subsequent pharmacokinetic studies may help us to understand these differences.
Various examples of ligands that exert divergent effects on CB1R signaling pathways have been described.Certain cannabinoids favor G protein-mediated signaling over the β-arrestin pathway, as in the case of novel compounds PNR-4-20 and PNR-4-02 that selectively activate the G αi pathway, while eliciting significantly less β-arrestin2 recruitment 36 .On the other hand, the allosteric modulator ORG27569 induces CB1R conformation state that selectively activates the ERK1/2 cascade via β-arrestin1 37 .Distinct ligands induce and stabilize different conformations of a given GPCR.Consequently, these conformations could preferentially activate a particular signaling cascade over others, a phenomenon called "biased signaling".Activation of a pathway resulting in desired therapeutic efficacy, together with a decrease of signaling pathways leading to undesired effects, typically psychoactivity or tolerance, may have profound consequences in drug discovery of molecules with potential medicinal uses, including those acting via CB1R.CB1R-mediated signaling is complex, and its outcome depends on the cellular environment, associated protein network, and ligand that activates the receptor in a particular way, or modulates its signaling in a unique way for each HHC enantiomer.
In neurons and other naïve cells, CB1R-interacting proteins also bias the signaling of the receptor, for example, SH3-containing GRB2-like protein 3-interacting protein 1 (SGIP1) 24,25,38,39 , Cannabinoid Receptor Interacting Protein 1a and 1b (CRIP1a/b) 40,41 , and G Protein-Coupled Receptor Associated Protein 1 (GASP1) 42,43 .The situation may become yet more complex with heterodimers of CB1R 44,45 .For example, CB1R was reported to form dimers with dopamine receptor 2. Activation of these heterodimeric receptors may activate the G αs pathway leading to the increase of cAMP, thus generating the opposite effect as when CB1R is signaling alone 44 .
The urgent need for better pharmaceutical management in patients prompts investigations for novel therapeutic agents.The ECS is involved in a plethora of nervous system physiology and pathophysiology.However, Figure 5. ERK1/2 phosphorylation elicited by the cannabinoids.HEK293 cells were transiently transfected with CB1R and empty vector (1:2 ratio).24 h after transfection, cells were stimulated by 10 µM of (9S)-HHC, (9R)-HHC, Δ 9 -THC or WIN, and kinetics of ERK1/2 phosphorylation were measured at the indicated times.The data are presented as means ± SEM of three independent experiments performed in 3 technical replicates.The statistical analysis is disclosed in Supplementary Table 6.*, p < 0.05 (9S)-HHC vs. WIN by ANOVA.implementing medical applications achieved by manipulating the ECS has been challenging, mainly due to the pleiotropic functions of the ECS.These include psychoactive and other undesired side effects of drugs acting on CB1R.Pharmacological approaches based on tinkering with the pleiotropic nature of CB1R signaling are one way to avoid undesired side effects.The biased CB1R-mediated signaling of the two HHC epimers, compared with each other and that of Δ 9 -THC (Fig. 6), together with the greater stability of HHC, represents an emerging prospective treatment via the ECS with possibly limited side effects.

Figure 1 .
Figure 1.The structures of the tested cannabinoids and the HHC synthesis scheme.(A) The structures of (9S)-HHC, (9R)-HHC, Δ 9 -THC, and WIN.(B) The synthesis of HHC from CBD, schematically via transformation of CBD into Δ 8 -THC and further reduction to obtain HHC epimers.

Figure 2 .
Figure 2. CB1R-driven G protein activation induced by the tested cannabinoids.HEK293 cells were transiently transfected with G αi1 -Rluc8 or G αoA -Rluc8, G β2 -Flag, G γ2 -VENUS, and SNAP-CB1R.The cells were stimulated with the indicated concentrations of (9S)-HHC, (9R)-HHC, Δ 9 -THC, WIN, or their vehicles.BRET donor and acceptor emission was measured 12 min after receptor stimulation.(A) Concentration-response relationship of G αi1 subunit dissociation from the G protein complex after CB1R stimulation.(B) Concentration-response relationship of G αoA subunit dissociation from the G protein complex after CB1R stimulation.The data are presented as means ± SEM from three independent experiments.The data analysis is disclosed in Supplementary Tables1 and 2.

Figure 4 .
Figure 4. Internalization of CB1R induced by the tested cannabinoids.Internalization was elicited by the application of 10 µM of (9S)-HHC, (9R)-HHC, Δ 9 -THC, and WIN.HEK293 cells were transiently transfected with the plasmids coding SNAP-CB1R or mock plasmid pRK6 (1:2 DNA mass ratio).Data represent net receptor internalization by each drug treatment (i.e.receptor internalization by the indicated drug minus receptor internalization by vehicle).The data are presented as means ± SEM of three independent experiments performed in 3 technical replicates.The statistical analysis is disclosed in Supplementary Table5.*, p < 0.05 by ANOVA.

Figure 6 .
Figure 6.Pharmacological profiles of (9S)-HHC, (9R)-HHC, and Δ 9 -THC.(A) Calculated maximum response values or the time-course peaks were plotted on the axes of the radar plot.For clathrin-mediated internalization (CME), the 60 min time points were used; for ERK1/2 phosphorylation, the 10 min time points were used.(B) Calculated maximum response values of the tested cannabinoids were represented as fractions of WIN and normalized to (9R)-HHC.The normalized values for β-arrestin interaction were plotted on the x-axis, and the means of the normalized values for G i1 /G oA activation were plotted on the y-axis.The values represent only the maximum responses elicited by the ligands.